Spontaneous release of acetylcholine from autonomic nerves in the bladder

Background and purpose:

Bladder contractility is regulated by intrinsic myogenic mechanisms interacting with autonomic nerves. In this study, we have investigated the physiological role of spontaneous release of acetylcholine in guinea pig and rat bladders.

Experimental approach:

Conventional isotonic or pressure transducers were used to record contractile activity of guinea pig and rat bladders.

Key results:

Hyoscine (3 µmol·L⁻¹), but not tetrodotoxin (TTX, 1 µmol·L⁻¹), reduced basal tension, distension-evoked contractile activity and physostigmine (1 µmol·L⁻¹)-evoked contractions of the whole guinea pig bladder and muscle strips in vitro. ω-Conotoxin GVIA (0.3 µmol·L⁻¹) did not affect physostigmine-induced contractions when given either alone or in combination with ω-agatoxin IVA (0.1 µmol·L⁻¹) and SNX 482 (0.3 µmol·L⁻¹). After 5 days in organotypic culture, when extrinsic nerves had significantly degenerated, the ability of physostigmine to induce contractions was reduced in the dorso-medial strips, but not in lateral strips (which have around 15 times more intramural neurones). Most muscle strips from adult rats lacked intramural neurones. After 5 days in culture, physostigmine-induced or electrical field stimulation-induced contractions of the rat bladder strips were greatly reduced. In anaesthetized rats, topical application of physostigmine (5–500 nmol) on the bladder produced a TTX-resistant tonic contraction that was abolished by atropine (4.4 µmol·kg⁻¹ i.v.).

Conclusions and implications:

The data indicate that there is spontaneous TTX-resistant release of acetylcholine from autonomic cholinergic extrinsic and intrinsic nerves, which significantly affects bladder contractility. This release is resistant to blockade of N, P/Q and R type Ca²⁺ channels.British Journal of Pharmacology (2009) 157, 607–619; doi:10.1111/j.1476-5381.2009.00166.x; published online 3 April 2009     

The omega-3 polyunsaturated fatty acid eicosapentaenoic acid inhibits mouse MC-26 colorectal cancer cell liver metastasis via inhibition of PGE2-dependent cell motility

BACKGROUND AND PURPOSE

The omega-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) has antineoplastic activity at early stages of colorectal carcinogenesis, relevant to chemoprevention of colorectal cancer (CRC). We tested the hypothesis that EPA also has anti-CRC activity at later stages of colorectal carcinogenesis, relevant to treatment of metastatic CRC, via modulation of E-type PG synthesis.

EXPERIMENTAL APPROACH

A BALB/c mouse model, in which intrasplenic injection of syngeneic MC-26 mouse CRC cells leads to development of liver metastases, was used. Dietary EPA was administered in the free fatty acid (FFA) form for 2 weeks before and after ultrasound-guided intrasplenic injection of 1 × 10⁶ MC-26 cells (n= 16 each group).

KEY RESULTS

Treatment with 5% (w w^-1) EPA-FFA was associated with a reduced MC-26 mouse CRC cell liver tumour burden compared with control animals (median liver weight 1.03 g vs. 1.62 g; P < 0.034). Administration of 5% EPA-FFA was also linked to a significant increase in tumour EPA incorporation and lower intratumoural PGE₂ levels (with concomitant increased production of PGE₃). Liver tumours from 5% EPA-FFA- treated mice demonstrated decreased 5-bromo-2-deoxyuridine-positive CRC cell proliferation and reduced phosphorylated ERK 1/2 expression at the invasive edge of tumours. A concentration-dependent reduction in MC-26 CRC cell Transwell® migration following EPA-FFA treatment (50–200 µM) in vitro was rescued by exogenous PGE₂ (10 µM) and PGE₁-alcohol (1 µM).

CONCLUSIONS AND IMPLICATIONS

EPA-FFA inhibits MC-26 CRC cell liver metastasis. EPA incorporation is associated with a ‘PGE₂ to PGE₃ switch’ in liver tumours. Inhibition of PGE₂-EP₄ receptor-dependent CRC cell motility probably contributes to the antineoplastic activity of EPA.     

KB-R7943, an inhibitor of the reverse Na+ /Ca2+ exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex I

BACKGROUND AND PURPOSE

An isothiourea derivative (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methane sulfonate (KB-R7943), a widely used inhibitor of the reverse Na⁺/Ca²⁺ exchanger (NCX_rev), was instrumental in establishing the role of NCX_rev in glutamate-induced Ca²⁺ deregulation in neurons. Here, the effects of KB-R7943 on N-methyl-D-aspartate (NMDA) receptors and mitochondrial complex I were tested.

EXPERIMENTAL APPROACH

Fluorescence microscopy, electrophysiological patch-clamp techniques and cellular respirometry with Seahorse XF24 analyzer were used with cultured hippocampal neurons; membrane potential imaging, respirometry and Ca²⁺ flux measurements were made in isolated rat brain mitochondria.

KEY RESULTS

KB-R7943 inhibited NCX_rev with IC₅₀= 5.7 ± 2.1 µM, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca²⁺ with IC₅₀= 13.4 ± 3.6 µM but accelerated calcium deregulation and mitochondrial depolarization in glutamate-treated neurons. KB-R7943 depolarized mitochondria in a Ca²⁺-independent manner. Stimulation of NMDA receptors caused NAD(P)H oxidation that was coupled or uncoupled from ATP synthesis depending on the presence of Ca²⁺ in the bath solution. KB-R7943, or rotenone, increased NAD(P)H autofluorescence under resting conditions and suppressed NAD(P)H oxidation following glutamate application. KB-R7943 inhibited 2,4-dinitrophenol-stimulated respiration of cultured neurons with IC₅₀= 11.4 ± 2.4 µM. With isolated brain mitochondria, KB-R7943 inhibited respiration, depolarized organelles and suppressed Ca²⁺ uptake when mitochondria oxidized complex I substrates but was ineffective when mitochondria were supplied with succinate, a complex II substrate.

CONCLUSIONS AND IMPLICATIONS

KB-R7943, in addition to NCX_rev, blocked NMDA receptors in cultured hippocampal neurons and inhibited complex I in the mitochondrial respiratory chain. These findings are critical for the correct interpretation of experimental results obtained with KB-R7943 and a better understanding of its neuroprotective action.     

Pentobarbital inhibition of human recombinant ��1A P/Q-type voltage-gated calcium channels involves slow, open channel block

BACKGROUND AND PURPOSE

Pre-synaptic neurotransmitter release is largely dependent on Ca²⁺ entry through P/Q-type (Ca_V2.1) voltage-gated Ca²⁺ channels (PQCCs) at most mammalian, central, fast synapses. Barbiturates are clinical depressants and inhibit pre-synaptic Ca²⁺ entry. PQCC barbiturate pharmacology is generally unclear, specifically in man. The pharmacology of the barbiturate pentobarbital (PB) in human recombinant α_1A PQCCs has been characterized.

EXPERIMENTAL APPROACH

PB effects on macroscopic Ca²⁺(I_Ca) and Ba²⁺(I_Ba) currents were studied using whole-cell patch clamp recording in HEK-293 cells heterologously expressing (α_1A)_human(β_2aα₂δ-1)_rabbit PQCCs.

KEY RESULTS

PB reversibly depressed peak current (I_peak) and enhanced apparent inactivation (fractional current at 800 ms, r₈₀₀) in a concentration-dependent fashion irrespective of charge carrier (50% inhibitory concentration: I_peak, 656 µM; r₈₀₀, 104 µM). Rate of mono-exponential I_Ba decay was linearly dependent on PB concentration. PB reduced channel availability by deepening non-steady-state inactivation curves without altering voltage dependence, slowed recovery from activity-induced unavailable states and produced use-dependent block. PB (100 µM) induced use-dependent block during physiological, high frequency pulse trains and overall depressed PQCC activity by two-fold.

CONCLUSION AND IMPLICATIONS

The results support a PB pharmacological mechanism involving a modulated receptor with preferential slow, bimolecular, open channel block (K_d = 15 µM). Clinical PB concentrations (<200 µM) inhibit PQCC during high frequency activation that reduces computed neurotransmitter release by 16-fold and is comparable to the magnitude of Ca²⁺-dependent facilitation, G-protein modulation and intrinsic inactivation that play critical roles in PQCC modulation underlying synaptic plasticity. The results are consistent with the hypothesis that PB inhibition of PQCCs contributes to central nervous system depression underlying anticonvulsant therapy and general anaesthesia.     

Role of connexin 43 in the maintenance of spontaneous activity in the guinea pig prostate gland

BACKGROUND AND PURPOSE

To investigate the role of connexin 43 in the maintenance of spontaneous activity in prostate tissue from young and old guinea pigs.

EXPERIMENTAL APPROACH

Conventional intracellular microelectrode and tension recording techniques, coupled with Western blot analysis and immunohistochemistry for connexin 43 (CX43) were used. The effects of three gap junction uncouplers, 18β glycyrrhetinic acid (10 µM, 40 µM), carbenoxolone (10 µM, 50 µM) and octanol (0.5 mM, 1 mM), were studied in cells displaying slow wave activity and on spontaneously contracting tissue from prostate glands of young (2–5 months) and old (9–16 months) guinea pigs.

KEY RESULTS

18β Glycyrrhetinic acid (40 µM), carbenoxolone (50 µM) or octanol (0.5 mM) abolished slow wave activity in prostate tissue from young and old guinea pigs and depolarized membrane potential by approximately 5 mV. These treatments also abolished all contractions in both sets of prostate tissue. These effects were reversed upon washout. Western blot analysis and CX43 immunohistochemistry showed that there was no age-related difference in the expression and distribution of CX43 in prostate tissues.

CONCLUSION AND IMPLICATIONS

When gap junctional communication via CX43 was disrupted, spontaneous activity was abolished at a cellular and whole tissue level; CX43 is therefore essential for the maintenance of spontaneous slow wave activity and subsequent contractile activity in the guinea pig prostate gland.     

Effects of rosiglitazone on the configuration of action potentials and ion currents in canine ventricular cells

BACKGROUND AND PURPOSE

In spite of its widespread clinical application, there is little information on the cellular cardiac effects of the antidiabetic drug rosiglitazone in larger experimental animals. In the present study therefore concentration-dependent effects of rosiglitazone on action potential morphology and the underlying ion currents were studied in dog hearts.

EXPERIMENTAL APPROACH

Standard microelectrode techniques, conventional whole cell patch clamp and action potential voltage clamp techniques were applied in enzymatically dispersed ventricular cells from dog hearts.

KEY RESULTS

At concentrations ≥10 µM rosiglitazone decreased the amplitude of phase-1 repolarization, reduced the maximum velocity of depolarization and caused depression of the plateau potential. These effects developed rapidly and were readily reversible upon washout. Rosiglitazone suppressed several transmembrane ion currents, concentration-dependently, under conventional voltage clamp conditions and altered their kinetic properties. The EC₅₀ value for this inhibition was 25.2 ± 2.7 µM for the transient outward K⁺ current (I_to), 72.3 ± 9.3 µM for the rapid delayed rectifier K⁺ current (I_Kr) and 82.5 ± 9.4 µM for the L-type Ca²⁺ current (I_Ca) with Hill coefficients close to unity. The inward rectifier K⁺ current (I_K1) was not affected by rosiglitazone up to concentrations of 100 µM. Suppression of I_to, I_Kr, and I_Ca was confirmed also under action potential voltage clamp conditions.

CONCLUSIONS AND IMPLICATIONS

Alterations in the densities and kinetic properties of ion currents may carry serious pro-arrhythmic risk in case of overdose with rosiglitazone, especially in patients having multiple cardiovascular risk factors, like elderly diabetic patients.

LINKED ARTICLE

This article is commented on by Hancox, pp. 496–498 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01281.x     

Role of T-type Ca2+ Channels in the Spontaneous Phasic Contraction of Pregnant Rat Uterine Smooth Muscle

Although extracellular Ca²⁺ entry through the voltage-dependent Ca²⁺ channels plays an important role in the spontaneous phasic contractions of the pregnant rat myometrium, the role of the T-type Ca²⁺ channels has yet to be fully identified. The aim of this study was to investigate the role of the T-type Ca²⁺ channel in the spontaneous phasic contractions of the rat myometrium. Spontaneous phasic contractions and [Ca²⁺]_i were measured simultaneously in the longitudinal strips of female Sprague-Dawley rats late in their pregnancy (on day 18~20 of gestation: term=22 days). The expression of T-type Ca²⁺ channel mRNAs or protein levels was measured. Cumulative addition of low concentrations (<1 µM) of nifedipine, a L-type Ca²⁺ channel blocker, produced a decrease in the amplitude of the spontaneous Ca²⁺ transients and contractions with no significant change in frequency. The mRNAs and proteins encoding two subunits (α1G, α1H) of the T-type Ca²⁺ channels were expressed in longitudinal muscle layer of rat myometrium. Cumulative addition of mibefradil, NNC 55-0396 or nickel induced a concentration-dependent inhibition of the amplitude and frequency of the spontaneous Ca²⁺ transients and contractions. Mibefradil, NNC 55-0396 or nickel also attenuated the slope of rising phase of spontaneous Ca²⁺ transients consistent with the reduction of the frequency. It is concluded that T-type Ca²⁺ channels are expressed in the pregnant rat myometrium and may play a key role for the regulation of the frequency of spontaneous phasic contractions.     

Inositol trisphosphate-dependent Ca2+ stores and mitochondria modulate slow wave activity arising from the smooth muscle cells of the guinea pig prostate gland

Background and purpose

Changes in smooth muscle tone of the prostate gland are involved in aetiology of symptomatic prostatic hyperplasia, however the control mechanisms of prostatic smooth muscle are not well understood. Here, we have examined the role of internal Ca²⁺ compartments in regulating slow wave activity in the guinea pig prostate.

Experimental approach

Standard intracellular membrane potential recording techniques were used.

Key results

The majority (89%) of impaled cells displayed ‘slow wave’ activity. Cyclopiazonic acid (10 µmol·L⁻¹) transiently depolarized (3–9 mV) the membrane potential of the prostatic stroma and transiently increased slow wave frequency. Thereafter, slow wave frequency slowly decreased over 20–30 min. Ryanodine transiently increased slow wave frequency, although after 30 min exposure slow wave frequency and time course returned to near control values. Caffeine (1 mmol·L⁻¹) reduced slow wave frequency, accompanied by membrane depolarization of about 8 mV. Blockade of inositol trisphosphate receptor (IP₃R)-mediated Ca²⁺ release with 2-aminoethoxy-diphenylborate (60 µmol·L⁻¹) or Xestospongin C (3 µmol·L⁻¹) or inhibiting phospholipase C and IP₃ formation using {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (5 µmol·L⁻¹) or neomycin (1 and 4 mmol·L⁻¹) reduced slow wave frequency, amplitude and duration. The mitochondrial uncouplers, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (1–10 µmol·L⁻¹), carbonyl cyanide m-chlorophenylhydrazone (1–3 µmol·L⁻¹) or rotenone (10 µmol·L⁻¹), depolarized the membrane (8–10 mV) before abolishing electrical activity.

Conclusion and implications

These results suggest that slow wave activity was dependent on the cyclical release of Ca²⁺ from IP₃-controlled internal stores and mitochondria. This implies that intracellular compartments were essential in the initiation and/or maintenance of the regenerative contractile activity in the guinea pig prostate gland.     

Inhibition of microsomal prostaglandin E2 synthase-1 as a molecular basis for the anti-inflammatory actions of boswellic acids from frankincense

BACKGROUND AND PURPOSE

Frankincense, the gum resin derived from Boswellia species, showed anti-inflammatory efficacy in animal models and in pilot clinical studies. Boswellic acids (BAs) are assumed to be responsible for these effects but their anti-inflammatory efficacy in vivo and their molecular modes of action are incompletely understood.

EXPERIMENTAL APPROACH

A protein fishing approach using immobilized BA and surface plasmon resonance (SPR) spectroscopy were used to reveal microsomal prostaglandin E₂ synthase-1 (mPGES1) as a BA-interacting protein. Cell-free and cell-based assays were applied to confirm the functional interference of BAs with mPGES1. Carrageenan-induced mouse paw oedema and rat pleurisy models were utilized to demonstrate the efficacy of defined BAs in vivo.

KEY RESULTS

Human mPGES1 from A549 cells or in vitro-translated human enzyme selectively bound to BA affinity matrices and SPR spectroscopy confirmed these interactions. BAs reversibly suppressed the transformation of prostaglandin (PG)H₂ to PGE₂ mediated by mPGES1 (IC₅₀ = 3–10 µM). Also, in intact A549 cells, BAs selectively inhibited PGE₂ generation and, in human whole blood, β-BA reduced lipopolysaccharide-induced PGE₂ biosynthesis without affecting formation of the COX-derived metabolites 6-keto PGF_1α and thromboxane B₂. Intraperitoneal or oral administration of β-BA (1 mg·kg⁻¹) suppressed rat pleurisy, accompanied by impaired levels of PGE₂ and β-BA (1 mg·kg⁻¹, given i.p.) also reduced mouse paw oedema, both induced by carrageenan.

CONCLUSIONS AND IMPLICATIONS

Suppression of PGE₂ formation by BAs via interference with mPGES1 contribute to the anti-inflammatory effectiveness of BAs and of frankincense, and may constitute a biochemical basis for their anti-inflammatory properties.     

The antipyretic effect of dipyrone is unrelated to inhibition of PGE(2) synthesis in the hypothalamus

BACKGROUND AND PURPOSE

Bacterial lipopolysaccharide (LPS) induces fever through two parallel pathways; one, prostaglandin (PG)-dependent and the other, PG-independent and involving endothelin-1 (ET-1). For a better understanding of the mechanisms by which dipyrone exerts antipyresis, we have investigated its effects on fever and changes in PGE₂ content in plasma, CSF and hypothalamus induced by either LPS or ET-1.

EXPERIMENTAL APPROACH

Rats were given (i.p.) dipyrone (120 mg·kg⁻¹) or indomethacin (2 mg·kg⁻¹) 30 min before injection of LPS (5 µg·kg⁻¹, i.v.) or ET-1 (1 pmol, i.c.v.). Rectal temperature was measured by tele-thermometry. PGE₂ levels were determined in the plasma, CSF and hypothalamus by elisa.

KEY RESULTS

LPS or ET-1 induced fever and increased CSF and hypothalamic PGE₂ levels. Two hours after LPS, indomethacin reduced CSF and hypothalamic PGE₂ but did not inhibit fever, while at 3 h it reduced all three parameters. Three hours after ET-1, indomethacin inhibited the increase in CSF and hypothalamic PGE₂ levels but did not affect fever. Dipyrone abolished both the fever and the increased CSF PGE₂ levels induced by LPS or ET-1 but did not affect the increased hypothalamic PGE₂ levels. Dipyrone also reduced the increase in the venous plasma PGE₂ concentration induced by LPS.

CONCLUSIONS AND IMPLICATIONS

These findings confirm that PGE₂ does not play a relevant role in ET-1-induced fever. They also demonstrate for the first time that the antipyretic effect of dipyrone was not mechanistically linked to the inhibition of hypothalamic PGE₂ synthesis.     

The NADPH oxidase inhibitor diphenyleneiodonium is also a potent inhibitor of cholinesterases and the internal Ca2+ pump

Background and purpose:

Diphenyleneiodonium (DPI) is often used as an NADPH oxidase inhibitor, but is increasingly being found to have unrelated side effects. We investigated its effects on smooth muscle contractions and the related mechanisms.

Experimental approach:

We studied isometric contractions in smooth muscle strips from bovine trachea. Cholinesterase activity was measured using a spectrophotometric assay; internal Ca²⁺ pump activity was assessed by Ca²⁺ uptake into smooth muscle microsomes.

Key results:

Contractions to acetylcholine were markedly enhanced by DPI (10⁻⁴ M), whereas those to carbachol (CCh) were not, suggesting a possible inhibition of cholinesterase. DPI markedly suppressed contractions evoked by CCh, KCl and 5-HT, and also unmasked phasic activity in otherwise sustained responses. Direct biochemical assays confirmed that DPI was a potent inhibitor of acetylcholinesterase and butyrylcholinesterase (IC₅₀∼8 × 10⁻⁶ M and 6 × 10⁻⁷ M, respectively), following a readily reversible, mixed non-competitive type of inhibition. The inhibitory effects of DPI on CCh contractions were not mimicked by another NADPH oxidase inhibitor (apocynin), nor the Src inhibitors PP1 or PP2, ruling out an action through the NADPH oxidase signalling pathway. Several features of the DPI-mediated suppression of agonist-evoked responses (i.e. suppression of peak magnitudes and unmasking of phasic activity) are similar to those of cyclopiazonic acid, an inhibitor of the internal Ca²⁺ pump. Direct measurement of microsomal Ca²⁺ uptake revealed that DPI modestly inhibits the internal Ca²⁺ pump.

Conclusions and implications:

DPI inhibits cholinesterase activity and the internal Ca²⁺ pump in tracheal smooth muscle.     

IL-1β reduces tonic contraction of mesenteric lymphatic muscle cells,with the involvement of cycloxygenase-2 and prostaglandin E2

Background and Purpose

The lymphatic system maintains tissue homeostasis by unidirectional lymph flow, maintained by tonic and phasic contractions within subunits, ‘lymphangions’. Here we have studied the effects of the inflammatory cytokine IL-1β on tonic contraction of rat mesenteric lymphatic muscle cells (RMLMC).

Experimental Approach

We measured IL-1β in colon-conditioned media (CM) from acute (AC-CM, dextran sodium sulfate) and chronic (CC-CM, T-cell transfer) colitis-induced mice and corresponding controls (Con-AC/CC-CM). We examined tonic contractility of RMLMC in response to CM, the cytokines h-IL-1β or h-TNF-α (5, 10, 20 ng·mL⁻¹), with or without COX inhibitors [TFAP (10⁻⁵ M), diclofenac (0.2 × 10⁻⁵ M)], PGE₂ (10⁻⁵ M)], IL-1-receptor antagonist, Anakinra (5 μg·mL⁻¹), or a selective prostanoid EP₄ receptor antagonist, GW627368X (10⁻⁶ and 10⁻⁷ M).

Key Results

Tonic contractility of RMLMC was reduced by AC- and CC-CM compared with corresponding control culture media, Con-AC/CC-CM. IL-1β or TNF-α was not found in Con-AC/CC-CM, but detected in AC- and CC-CM. h-IL-1β concentration-dependently decreased RMLMC contractility, whereas h-TNF-α showed no effect. Anakinra blocked h-IL-1β-induced RMLMC relaxation, and with AC-CM, restored contractility to RMLMC. IL-1β increased COX-2 protein and PGE₂ production in RMLMC.. PGE₂ induced relaxations in RMLMC, comparable to h-IL-1β. Conversely, COX-2 and EP₄ receptor inhibition reversed relaxation induced by IL-1β.

Conclusions and Implications

The IL-1β-induced decrease in RMLMC tonic contraction was COX-2 dependent, and mediated by PGE₂. In experimental colitis, IL-1β and tonic lymphatic contractility were causally related, as this cytokine was critical for the relaxation induced by AC-CM and pharmacological blockade of IL-1β restored tonic contraction.     

Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: evidence of a role for L-type voltage-operated Ca2+ channels

BACKGROUND AND PURPOSE

Diabetic cystopathy is one of the most common and incapacitating complications of diabetes mellitus. This study aimed to evaluate the functional, structural and molecular alterations of detrusor smooth muscle (DSM) in streptozotocin-induced diabetic mice, focusing on the contribution of Ca²⁺ influx through L-type voltage-operated Ca²⁺ channels (L-VOCC).

EXPERIMENTAL APPROACH

Male C57BL/6 mice were injected with streptozotocin (125 mg·kg⁻¹). Four weeks later, contractile responses to carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation were measured in urothelium-intact DSM strips. Cystometry and histomorphometry were performed, and mRNA expression for muscarinic M₂/M₃ receptors, purine P2X1 receptors and L-VOCC in the bladder was determined.

KEY RESULTS

Diabetic mice exhibited higher bladder capacity, frequency, non-void contractions and post-void pressure. Increased bladder weight, wall thickness, bladder volume and neural tissue were observed in diabetic bladders. Carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation all produced greater DSM contractions in diabetic mice. The L-VOCC blocker nifedipine almost completely reversed the enhanced DSM contractions in bladders from diabetic animals. The Rho-kinase inhibitor Y27632 had no effect on the enhanced carbachol contractions in the diabetic group. Expression of mRNA for muscarinic M₃ receptors and L-VOCC were greater in the bladders of diabetic mice, whereas levels of M₂ and P2X1 receptors remained unchanged.

CONCLUSIONS AND IMPLICATIONS

Diabetic mice exhibit features of urinary bladder dysfunction, as characterized by overactive DSM and decreased voiding efficiency. Functional and molecular data suggest that overactive DSM in diabetes is the result of enhanced extracellular Ca²⁺ influx through L-VOCC.     

Piceatannol, a derivative of resveratrol, moderately slows INa inactivation and exerts antiarrhythmic action in ischaemia-reperfused rat hearts

Background and purpose:

Piceatannol is more potent than resveratrol in free radical scavenging in association with antiarrhythmic and cardioprotective activities in ischaemic-reperfused rat hearts. The present study aimed to investigate the antiarrhythmic efficacy and the underlying ionic mechanisms of piceatannol in rat hearts.

Experimental approach:

Action potentials and membrane currents were recorded by the whole-cell patch clamp techniques. Fluo-3 fluorimetry was used to measure cellular Ca²⁺ transients. Antiarrhythmic activity was examined from isolated Langendorff-perfused rat hearts.

Key results:

In rat ventricular cells, piceatannol (3–30 µmol·L⁻¹) prolonged the action potential durations (APDs) and decreased the maximal rate of upstroke (V_max) without altering Ca²⁺ transients. Piceatannol decreased peak I_Na and slowed I_Na inactivation, rather than induced a persistent non-inactivating current, which could be reverted by lidocaine. Resveratrol (100 µmol·L⁻¹) decreased peak I_Na without slowing I_Na inactivation. The inhibition of peak I_Na or V_max was associated with a negative shift of the voltage-dependent steady-state I_Na inactivation curve without altering the activation threshold. At the concentrations more than 30 µmol·L⁻¹, piceatannol could inhibit I_Ca,L, I_to, I_Kr, Ca²⁺ transients and Na⁺-Ca²⁺ exchange except I_K1. Piceatannol (1–10 µmol·L⁻¹) exerted antiarrhythmic activity in isolated rat hearts subjected to ischaemia-reperfusion injury.

Conclusions and implications:

The additional hydroxyl group on resveratrol makes piceatannol possessing more potent in I_Na inhibition and uniquely slowing I_Na inactivation, which may contribute to its antiarrhythmic actions at low concentrations less than 10 µmol·L⁻¹.     

Vascular actions of calcimimetics: role of Ca²(+) -sensing receptors versus Ca²(+) influx through L-type Ca²(+) channels

BACKGROUND AND PURPOSE

The calcimimetic, (R)-N-(3-(3-(trifluoromethyl)phenyl)propyl)-1-(1-napthyl)ethylamine hydrochloride (cinacalcet), which activates Ca²⁺-sensing receptors (CaR) in parathyroid glands, is used to treat hyperparathyroidism. Interestingly, CaR in perivascular nerves or endothelial cells is also thought to modulate vascular tone. This study aims to characterize the vascular actions of calcimimetics.

EXPERIMENTAL APPROACH

In rat isolated small mesenteric arteries, the relaxant responses to the calcimimetics, cinacalcet and (R)-2-[[[1-(1-naphthyl)ethyl]amino]methyl]-1H-indole hydrochloride (calindol) were characterized, with particular emphasis on the role of CaR, endothelium, perivascular nerves, K⁺ channels and Ca²⁺ channels. Effects of L-ornithine, which activates a Ca²⁺-sensitive receptor related to CaR (GPRC6A), were also tested.

KEY RESULTS

Cinacalcet induced endothelium-independent relaxation (pEC₅₀ 5.58 ± 0.07, E_max 97 ± 6%) that was insensitive to sensory nerve desensitization by capsaicin or blockade of large-conductance Ca²⁺-activated K⁺ channels by iberiotoxin. Calindol, another calcimimetic, caused more potent relaxation (pEC₅₀ 6.10 ± 0.10, E_max 101 ± 6%), which was attenuated by endothelial removal or capsaicin, but not iberiotoxin. The negative modulator of CaR, calhex 231 or changes in [Ca²⁺]_o had negligible effect on relaxation to both calcimimetics. The calcimimetics relaxed vessels precontracted with high [K⁺]_o and inhibited Ca²⁺ influx in endothelium-denuded vessels stimulated by methoxamine, but not ionomycin. They also inhibited contractions to the L-type Ca²⁺ channel activator, BayK8644. L-ornithine induced small relaxation alone and had no effect on the responses to calcimimetics.

CONCLUSION AND IMPLICATIONS

Cinacalcet and calindol are potent arterial relaxants. Under the experimental conditions used, they predominantly act by inhibiting Ca²⁺ influx through L-type Ca²⁺ channels into vascular smooth muscle, whereas Ca²⁺-sensitive receptors (CaR or GPRC6A) play a minor role.     

Interaction of diltiazem with an intracellularly accessible binding site on Ca(V)1.2

BACKGROUND AND PURPOSE

Diltiazem inhibits Ca_V1.2 channels and is widely used in clinical practice to treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α₁ subunit of Ca_V1.2. The aim of the present study was to clarify the location of the diltiazem binding site making use of its membrane-impermeable quaternary derivative d-cis-diltiazem (qDil) and mutant α₁ subunits.

EXPERIMENTAL APPROACH

Ca_V1.2 composed of α1, α2-δ and β2a subunits were expressed in tsA-201 cells and barium currents through Ca_V1.2 channels were recorded using the patch clamp method in the whole cell configuration. qDil was synthesized and applied to the intracellular side (via the patch pipette) or to the extracellular side of the membrane (by bath perfusion).

KEY RESULTS

Quaternary derivative d-cis-diltiazem inhibited Ca_V1.2 when applied to the intracellular side of the membrane in a use-dependent manner (59 ± 4% at 300 µM) and induced only a low level of tonic (non-use-dependent) block (16 ± 2% at 300 µM) when applied to the extracellular side of the membrane. Mutations in IIIS6 and IVS6 that have previously been shown to reduce the sensitivity of Ca_V1.2 to tertiary diltiazem also had reduced sensitivity to intracellularly applied qDil.

CONCLUSION AND IMPLICATIONS

The data show that use-dependent block of in Ca_V1.2 by diltiazem occurs by interaction with a binding site accessible via a hydrophilic route from the intracellular side of the membrane.     

The flavonoid scaffold as a template for the design of modulators of the vascular Ca(v) 1.2 channels

BACKGROUND AND PURPOSE

Previous studies have pointed to the plant flavonoids myricetin and quercetin as two structurally related stimulators of vascular Ca_v1.2 channel current (I_Ca1.2). Here we have tested the proposition that the flavonoid structure confers the ability to modulate Ca_v1.2 channels.

EXPERIMENTAL APPROACH

Twenty-four flavonoids were analysed for their effects on I_Ca1.2 in rat tail artery myocytes, using the whole-cell patch-clamp method.

KEY RESULTS

Most of the flavonoids stimulated or inhibited I_Ca1.2 in a concentration- and voltage-dependent manner with EC₅₀ values ranging between 4.4 µM (kaempferol) and 16.0 µM (myricetin) for the stimulators and IC₅₀ values between 13.4 µM (galangin) and 100 µM [(±)-naringenin] for the inhibitors. Key structural requirements for I_Ca1.2 stimulatory activity were the double bond between C2 and C3 and the hydroxylation pattern on the flavonoid scaffold, the latter also determining the molecular charge, as shown by molecular modelling techniques. Absence of OH groups in the B ring was key in I_Ca1.2 inhibition. The functional interaction between quercetin and either the stimulator myricetin or the antagonists resokaempferol, crysin, genistein, and 5,7,2′-trihydroxyflavone revealed that quercetin expressed the highest apparent affinity, in the low µM range, for Ca_v1.2 channels. Neither protein tyrosine kinase nor protein kinase Cα were involved in quercetin-induced stimulation of I_Ca1.2.

CONCLUSIONS AND IMPLICATIONS

Quercetin-like plant flavonoids were active on vascular Ca_v1.2 channels. Thus, the flavonoid scaffold may be a template for the design of novel modulators of vascular smooth muscle Ca_v1.2 channels, valuable for the treatment of hypertension and stroke.     

A Ca²⁺-dependent chloride current and Ca²⁺ influx via Ca(v)1.2 ion channels play major roles in P2Y receptor-mediated pulmonary vasoconstriction

BACKGROUND AND PURPOSE

ATP, UTP and UDP act at smooth muscle P2X and P2Y receptors to constrict rat intrapulmonary arteries, but the underlying signalling pathways are poorly understood. Here, we determined the roles of the Ca²⁺-dependent chloride ion current (I_Cl,Ca), Ca_v1.2 ion channels and Ca²⁺ influx.

EXPERIMENTAL APPROACH

Isometric tension was recorded from endothelium-denuded rat intrapulmonary artery rings (i.d. 200–500 µm) mounted on a wire myograph.

KEY RESULTS

The I_Cl,Ca blockers, niflumic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and the Ca_v1.2 channel blocker, nifedipine, reduced peak amplitude of contractions evoked by UTP and UDP by ∼45–50% and in a non-additive manner. Ca²⁺-free buffer inhibited responses by ∼70%. Niflumic acid and nifedipine similarly depressed contractions to ATP, but Ca²⁺-free buffer almost abolished the response. After peaking, contractions to UTP and UDP decayed slowly by 50–70% to a sustained plateau, which was rapidly inhibited by niflumic acid and nifedipine. Contractions to ATP, however, reversed rapidly and fully. Tannic acid contracted tissues per se and potentiated nucleotide-evoked contractions.

CONCLUSIONS AND IMPLICATIONS

I_Cl,Ca and Ca²⁺ influx via Ca_v1.2 ion channels contribute substantially and equally to contractions of rat intrapulmonary arteries evoked by UTP and UDP, via P2Y receptors. ATP also activates these mechanisms via P2Y receptors, but the greater dependence on extracellular Ca²⁺ most likely reflects additional influx through the P2X1 receptor pore. The lack of a sustained response to ATP is probably due to it acting at P2 receptor subtypes that desensitize rapidly. Thus multiple signalling mechanisms contribute to pulmonary artery vasoconstriction mediated by P2 receptors.